1. Field of Invention
The present invention generally relates to a liver-assist device and liver cell clones which may find application in artificial liver systems, extracorporeal liver-assist devices and bioreactors. More particularly, the present invention is directed to a liver cell clone with enhanced detoxification activities. The liver cell clone is characterized as having increased GSH content and elevated GST activity as compared to conventional immortalized human hepatocyte cell lines and primary human hepatocyte cultures isolated from liver tissue.
2. Background of the Related Art
Over the past several years, the focus of investigations employing extracorporeal liver support systems for the treatment of fulminant liver failure and liver dysfunction has been shifting from basic experimentation and animal trials to clinical evaluations and applications. Some groups in the United States are conducting, under the supervision of the Food and Drug Administration, clinical trials with various bioartificial liver devices (See, e.g., Rozga J. et al., J. Ann. Surg. 219:538-46, 1994, Sussman N. L. et al., Amer. J. Kid. Dis. 18:371-84, 1996, Sussman N. L., Clin Invest. Med. 19:393-9, 1996, Dixit V., Scan. J. Gastroenterol. Suppl. 220:101-14, 1996). Similarly, several groups in Euro-Asia (in particular, Germany, the Netherlands, Great Britain, and Russia) have commenced pre-clinical trials into the effectiveness of bioartificial-liver devices in treating liver dysfunctions (See, e.g., Gerlach J., Transplant. Proc. 29:852, 1997, Hughes R. D et al., Semin. Liver Dis. 16:435-444, 1996). Research into bioartificial liver support devices is also quite active in Japan.
The immediate objective of temporary liver support (which may be provided by bioartifical-liver devices) is to maintain a patient with acute or fulminant hepatic failure or dysfunction until the patient's own liver regenerates. From the clinical data available, it appears that liver support systems are mainly used to bridge the patient to orthotopic transplantation, to improve cerebral circulation and to improve hemo-dynamic parameters.
Rozga and his group have reported on 19 patients treated with bioartificial liver (BAL) devices for 7-hour periods on one to five occasions. Two groups of patients were studied, eleven (11) with acute liver failure and eight (8) with chronic liver failure. Liver transplantation was performed at a mean of 39 hours after initiating BAL support. Thirteen (13) patients, all eleven (11) suffering from acute liver failure and two (2) of the chronic liver failure group, survived after liver transplantation. Patients who were not candidates for transplantation, and therefore did not receive a liver transplant, died (Kamlot A. and Rozga J., Biotech & Bioeng. 50:382-91, 1996).
In a clinical study carried out by Sussman et al. (Sussman N. L. et al., Amer. J. Kid. Dis. 18:371-84, 1996), eleven (11) patients with acute liver failure were treated with an extracorporeal liver assist device (ELAD) for up to 114 hours. Nine of ten patients in whom multiple estimations of galactose elimination capacity were measured showed improved galactose elimination capacity. One (1) patient recovered without transplantation, four (4) patients underwent transplantation, and the others died.
Only occasionally has treatment with bioartificial liver support systems, ELAD or BAL, alone been shown to lead to complete recovery (Sheil A. G. R. et al., Aust. N. Z. J. Surg. 66:547-52, 1996; Watanabe F. D. et al., Ann. Surg. 225:484-91, 1997). Further, although hepatocyte-based hybrid extracorporeal livier support devices have demonstrated significant improvement in metabolic functions in a variety of experimental and animal tests, such degree of improvement has been difficult to duplicate completely in humans. The efficiency of treatment demonstrated in human patients suffering from hepatic failure is not so striking as compared to the efficiency seen in animals, as shown in those patients who are treated with either of the two most widely used extracorporeal liver-assist systems. For example, a well-controlled clinical trial recently carried out in the U.K to assess the overall value of ELAD (Sussmann) and BAL (Rozga) in the treatment of patients suffering from hepatic dysfunction, showed that while the two systems enhanced certain metabolic functions in such patients, the magnitude of the enhancement was not at a desired level (Hughes R. D. et al., Semin. Liver Dis. 16:435-444, 1996). Similar studies suggest that the functional capacity of conventional extracoporeal liver-assist devices may be limited, particularly considering the number of metabolic functions needing correction in the fulminant patient.
Numerous obstacles have impeded the development of efficient extracorporeal liver-support devices for routine clinical application. These are: (1) an imperfect understanding of the basic pathophysiology of liver dysfunction, liver biology and the regulation of the proliferation of hepatocytes; (2) absence of knowledge in regard to the mechanisms involved in the development of hepatic coma and/or encephalopathy in fulminant hepatic failure; (3) an inability to produce, and maintain in a bioreactor, a sufficient number of hepatocytes with sufficient. bio-activity to correct failing liver functions in fulminant hepatic failure patients. At present, hepatocytes used in conventional extracorporeal liver-assist devices are typically harvested from primary liver cultures obtained from animals, particularly the pig, or consist of immortalized cells derived from humans. Fresh hepatocytes have the advantage of differentiated functionality, but lack proliferation ability. The situation is Just reverse in term of immortalized cells. These two types of hepatocytes are often at opposite ends of a biologic continuum.
As differentiation and proliferation are found to be incompatible in a large number of cell types (evidently in accord with some basic biological principle), it is often difficult to obtain large number of hepatocytes with desired multiple functionalities. Common BAL devices contain about 50 g primary cultures from pig liver, which is significantly lower than that physiologically minimal mass (ranging from 200-400 g) hypothesized to be needed for efficient metabolic activity. On the other hand, even ELAD devices employing as much as 400 g of C3A immortalized cells have provided insufficient major synthetic and metabolic functions (Hughes R D et al, Liver Transpl Surg 1:200-6, 1995). For example, Gailetti et al. conducted a study which ostensibly used adequate amounts of primary hepatocyte cultures in a complex and well-designed configuration, desirable function could only be found for a limited period of about 7 hours (Gailetti P. M. & Jauregui H. O., in Handbook of Bioengineering pp 1952-66, 1995).
At this time, continuous support of patients with fulminant hepatic failure appears to be impractical using fresh pig livers. Further, no method has been developed yet which adequately prevents hepatocytes from a human or porcine source from deteriorating in vitro. Although great efforts have been made to improve the performance of hepatocytes in such systems, including primary culture from xenograft and established cell lines, no report has claimed to maintain desired functions of differentiated hepatocytes for significant periods of time in bioartificial liver-support system bioreactors. Loss of hepatic functions in culture appears to be associated with cellular responses involving cellular interactions and cellular apoptosis in vitro (Collins B. H. et al., Transplantation 58:1162-71, 1994); Rivern D. J. et al. Transplant Proc. 31:671-73, 1999). It should be noted that cells isolated from the body are maintained in a non-physiologic environment and are cut off from numerous regulatory mechanisms which control hepatocyte function in vivo. It appears that the hepatocyte bioreactors for bioartificial liver support have reached their maximal performance at present, and that further improvement will not be seen unless there are theoretical and technological breakthroughs made to harvest multiple functional hepatocytes.
On the whole, it may be argued that bioartificial liver systems fail to show a significant advantage over totally artificial liver systems. Some artificial liver systems, such as those based on modern membrane technology and absorbents, have already been the subject of positive clinical assessment (Ash S. R. et al., Intl. J. Artif. Org. 15:151-61, 1992). For example, highly adsorptive dialysis membrane coated with albumin has been reported to facilitate the removal of toxins (Stange J. et al., Artif. Org. 17:809-13, 1993). As BAL systems conventionally use traditional charcoal adsorbents in the plasma circuit before the hepatocyte bioreactor (Rozga J. & Demetriou A. A., ASAIO J. 41:831-37, 1995), it is difficult to assess how much of the effect seen with BAL treatment is due to the charcoal, and how much due to the hepatocytes in the bioreactor.
It is also important to sort out the priority in which toxins should be removed. In general, both total artificial and bioartificial liver support systems have been designed to remove a dozen or so potential toxins that are recognized as factors causing encephalopathy in hepatic failure patients. Removal of the toxins is generally accomplished by use of adsorbents, dialysis, and viable hepatocytes. Unfortunately in the clinic, however, patient outcome has not always been found to be proportional to the circulating level of any these toxins, such as ammonia, imbalanced amino acids. It remains uncertain whether hepatic coma is due to any one toxin, a synergistic effect between toxins, or other factor.
In hepatic failure, the toxic substances come from two sources. They enter into the systemic circulation because of failure of the liver to clear substances from portal blood. Toxins also accumulate in a patient's circulation as result of necrosis of the liver tissue itself. Less is known about the nature and role of the necrotic substances in the development of encephalopathy than with respect to the toxins that are not cleared by the dysfunctional liver. There is, however, some indirect evidence implicating the importance of these necrotic substances. For example, it is known that when necrotic tissue is removed from patient body, i.e, hepatectomy is carried out before transplantation, improvement in systemic hemodynamic parameters (systemic vascular resistance and oxygen utilization) and reduction in intracranial pressure (ICP) can be achieved (Ringe B. et al., Transplant Proc. 20:552-7, 1988; Harrison P M et al., Gut 32:A837, 1991). Patients receiving orthotopic liver transplantation after treatment with extracorporeal liver support fare significantly better than patients not receiving transplantation, presumably because the necrotic liver in such patients is not removed.
Necrotic tissue can release some toxic metabolites that include a considerable amount of reactive oxygen substance species. Oxidative stress results in mitochondrial dysfunction, membrane injury and denaturation of DNA and other cell components (Sewell R. B. et al., Clin. Sci. 63:237, 1982). Excessive production of oxygen radicals may lead to altered enzymatic activities, decreased DNA repair, impaired utilization of oxygen, glutathione depletion and lipid peroxidation. The circulating toxins can cause pathological changes such as inflammatory reactions in all part of body, but in particular cerebral cells that are quite sensitive to these toxins. Damage to the cerebral cells may result in cerebral edema featured by increased intracranial pressure. Failure to obtain proper treatment may lead to other complications such as multiple organ failure (MOF), onset of severe clotting abnormalities, renal failure, pulmonary edema or cardiovascular collapse. It has been hypothesized that encepchalopathy is an early symptom of multiple organ failure caused by toxins released from necrotic liver tissue. There are also a few reports that indicate the involvement of active oxygen species toxins from necrotic tissue in the development of hepatic coma. As eradication of necrotic toxins might be an alternative to bioartificial liver support, such eradication may play an increasing role in the future design of bioreactors for liver support systems.
It has been suggested that an extracorporeal artificial liver support system does not necessarily require that hepatocytes used be able to replace all missing bio-functionality, and that some specific functional activities that happen to be missing or deficient in the patients might be ameliorated in an alternative manner.
Acute liver failure is usually associated with hepatic encephalopathy, which results in high mortality. The main purpose of an artificial liver-support system is to prevent the patient suffering from fulminant hepatic failure from developing encephalopathy, or to treat encephalopathy if it has already developed. The reversible and global nature of hepatic encephalopathy strongly indicates that a metabolic abnormality rather than direct toxic injury is involved in its pathogenesis. There is also a great deal of evidence to demonstrate that many toxins accumulate in a patient's circulation upon hepatic failure, and that such toxins may be responsible for the initiation of encephalopathy. Therefore, detoxification or removal of toxins from patients suffering from fulminant hepatic failure is considered to be a primary goal of extracorporeal liver-assist devices. Previous studies on the removal of toxins from the blood of patients using absorbents or dialysis have not demonstrated significant improvement in the survival of patient's suffering from severe encephalopathy. The present inventors hypothesize that blood purification needs to become more specific and selective in order to provide a useful remedy for reducing the severity of hepatic encephalopathy.